Eiki Ichihara

AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer

UNLABELLED First-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provide significant clinical benefit in patients with advanced EGFR-mutant (EGFRm(+)) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent, and selective third-generation irreversible inhibitor of both EGFRm(+) sensitizing and T790M resistance mutants that spares wild-type EGFR. This mono-anilino-pyrimidine compound is structurally distinct from other third-generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Preclinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm(+) and EGFRm(+)/T790M(+) mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR-mutant tumor xenograft and transgenic models. The treatment of 2 patients with advanced EGFRm(+) T790M(+) NSCLC is described as proof of principle. SIGNIFICANCE We report the development of a novel structurally distinct third-generation EGFR TKI, AZD9291, that irreversibly and selectively targets both sensitizing and resistant T790M(+) mutant EGFR while harboring less activity toward wild-type EGFR. AZD9291 is showing promising responses in a phase I trial even at the first-dose level, with first published clinical proof-of-principle validation being presented.

Acquired Resistance to EGFR Inhibitors Is Associated with a Manifestation of Stem Cell–like Properties in Cancer Cells

Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI) is a critical problem in the treatment of lung cancer. Although several mechanisms have been shown to be responsible for acquired resistance, all mechanisms have not been uncovered. In this study, we investigated the molecular and cellular profiles of the acquired resistant cells to EGFR-TKI in EGFR-mutant lung cancers. Four EGFR-mutant cell lines were exposed to gefitinib by stepwise escalation and high-concentration exposure methods, and resistant sublines to gefitinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Although previously reported, alterations including secondary EGFR T790M mutation, MET amplification, and appearance of epithelial-to-mesenchymal transition (EMT) features were observed, these 2 drug-exposure methods revealed different resistance mechanisms. The resistant cells with EMT features exhibited downregulation of miRNA-200c by DNA methylation. Furthermore, the HCC827-derived subline characterized by the high-concentration exposure method exhibited not only EMT features but also stem cell-like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) overexpression, increase of side-population, and self-renewal capability. Resistant sublines with stem cell-like properties were resistant to conventional chemotherapeutic agents but equally sensitive to histone deacetylase and proteasome inhibitors, compared with their parental cells. ALDH1A1 was upregulated in clinical samples with acquired resistance to gefitinib. In conclusion, our study indicates that the manner of EGFR-TKI exposure influences the mechanism of acquired resistance and the appearance of stem cell-like property with EGFR-TKI treatment.

Acquired Resistance to the Mutant-Selective EGFR Inhibitor AZD9291 Is Associated with Increased Dependence on RAS Signaling in Preclinical Models

Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition.

RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer

One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS–mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRASWT) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK–positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes.

Liposomal Delivery of MicroRNA-7–Expressing Plasmid Overcomes Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor-Resistance in Lung Cancer Cells

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have been strikingly effective in lung cancers harboring activating EGFR mutations. Unfortunately, the cancer cells eventually acquire resistance to EGFR-TKI. Approximately 50% of the acquired resistance involves a secondary T790M mutation. To overcome the resistance, we focused on EGFR suppression using microRNA-7 (miR-7), targeting multiple sites in the 3′-untranslated region of EGFR mRNA. Two EGFR-TKI–sensitive cell lines (PC-9 and H3255) and two EGFR-TKI–resistant cell lines harboring T790M (RPC-9 and H1975) were used. We constructed miR-7-2 containing miR-7–expressing plasmid. After transfection of the miR-7–expressing plasmid, using cationic liposomes, a quantitative PCR and dual luciferase assay were conducted to examine the efficacy. The antiproliferative effect was evaluated using a cell count assay and xenograft model. Protein expression was examined by Western blotting. The miR-7 expression level of the transfectants was approximately 30-fold higher, and the luciferase activity was ablated by 92%. miR-7 significantly inhibited cell growth not only in PC-9 and H3255 but also in RPC-9 and H1975. Expression of insulin receptor substrate-1 (IRS-1), RAF-1, and EGFR was suppressed in the four cell lines. Injection of the miR-7–expressing plasmid revealed marked tumor regression in a mouse xenograft model using RPC-9 and H1975. EGFR, RAF-1, and IRS-1 were suppressed in the residual tumors. These findings indicate promising therapeutic applications of miR-7–expressing plasmids against EGFR oncogene–addicted lung cancers including T790M resistance by liposomal delivery. Mol Cancer Ther; 10(9); 1720–7. ©2011 AACR.

Non–Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases

Crizotinib is the standard of care for advanced non-small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult.

Role of Survival Post-Progression in Phase III Trials of Systemic Chemotherapy in Advanced Non-Small-Cell Lung Cancer: A Systematic Review

Background In advanced non-small-cell lung cancer (NSCLC), with the increasing number of active compounds available in salvage settings, survival after progression to first-line chemotherapy seems to have improved. A literature survey was conducted to examine whether survival post-progression (SPP) has improved over the years and to what degree SPP correlates with overall survival (OS). Methods and Findings Median progression-free survival (MPFS) time and median survival time (MST) were extracted in phase III trials of first-line chemotherapy for advanced NSCLC. SPP was pragmatically defined as the time interval of MST minus MPFS. The relationship between MPFS and MST was modeled in a linear function. We used the coefficient of determination (r 2) to assess the correlation between them. Seventy trials with 145 chemotherapy arms were identified. Overall, median SPP was 4.7 months, and a steady improvement in SPP was observed over the 20 years (9.414-day increase per year; p<0.001) in parallel to the increase in MST (11.253-day increase per year; p<0.001); MPFS improved little (1.863-day increase per year). Overall, a stronger association was observed between MST and SPP (r 2 = 0.8917) than MST and MPFS time (r 2 = 0.2563), suggesting SPP and MPFS could account for 89% and 25% of the variation in MST, respectively. The association between MST and SPP became closer over the years (r 2 = 0.4428, 0.7242, and 0.9081 in 1988–1994, 1995–2001, and 2002–2007, respectively). Conclusions SPP has become more closely associated with OS, potentially because of intensive post-study treatments. Even in advanced NSCLC, a PFS advantage is unlikely to be associated with an OS advantage any longer due to this increasing impact of SPP on OS, and that the prolongation of SPP might limit the original role of OS for assessing true efficacy derived from early-line chemotherapy in future clinical trials.

Effects of Vandetanib on Lung Adenocarcinoma Cells Harboring Epidermal Growth Factor Receptor T790M Mutation In vivo

Vandetanib is a novel multitarget tyrosine kinase inhibitor (TKI) that inhibits vascular endothelial growth factor receptor-2 (VEGFR-2), with additional inhibition of epidermal growth factor receptor (EGFR) and rearranged during transfection receptor signaling, which has shown promising results in clinical trials for advanced non-small cell lung cancer. However, the mechanisms of acquired resistance to vandetanib remain unclear. Therefore, we established in vitro vandetanib-resistant PC-9/VanR cells from PC-9, a vandetanib-sensitive lung adenocarcinoma cell line, by chronic exposure to this agent. PC-9/VanR cells were 50-fold more resistant to vandetanib than PC-9 cells in vitro. Compared with PC-9 cells, PC-9/VanR cells showed emergence of an EGFR T790M mutation, moderately elevated MET amplification, and similar VEGFR-2 inhibition by vandetanib. Note that phospho-MET in PC-9/VanR was suppressed following EGFR inhibition by an irreversible EGFR-TKI, indicating that MET signaling of PC-9/VanR was dependent on EGFR signaling and that MET amplification was not the primary mechanism of resistance to vandetanib. In contrast to the in vitro experiment, vandetanib effectively inhibited the growth of PC-9/VanR tumors in an in vivo xenograft model through the antiangiogenesis effects of VEGFR-2 inhibition. In conclusion, the multitarget TKI vandetanib induced or selected for the EGFR T790M mutation as observed previously with highly selective EGFR-TKIs. However, vandetanib retained significant efficacy in vivo against xenografts harboring the T790M mutation, providing a strong scientific rationale for investigating vandetanib in clinical settings where acquired resistance through emergence of EGFR T790M mutations limits the effectiveness of highly selective EGFR-TKIs.

Phase II Trial of Gefitinib in Combination with Bevacizumab as First-Line Therapy for Advanced Non–Small Cell Lung Cancer with Activating EGFR Gene Mutations: The Okayama Lung Cancer Study Group Trial 1001

Purpose: Whether bevacizumab enhances the effect of the epidermal growth factor receptor (EGFR) inhibitor gefitinib on EGFR mutant non–small cell lung cancers (NSCLCs) remains unknown. We conducted a phase II trial to investigate the efficacy and safety of gefitinib when combined with bevacizumab as first-line therapy in patients with advanced NSCLC harboring EGFR gene mutations. Methods: In this trial, 42 patients with a performance status of 0 to 2 received gefitinib (250 mg/d) and bevacizumab (15 mg/kg, every 3 weeks). The primary end point of this study was the 1-year progression-free survival (PFS) rate. We assumed that a 1-year PFS rate of 55% would indicate potential usefulness and that a 1-year PFS rate of 40% would constitute the lower limit of interest. Results: Forty-two patients were enrolled in the study with a median age of 73 (range 42–86) years. Activating EGFR gene mutations included exon 19 deletion (57%) and L858R point mutations in exon 21 (38%). The objective response rate was 73.8% and included two complete responses. The 1-year PFS rate and median PFS time were 56.7% (95% confidence interval [CI] 39.9–70.5) and 14.4 months (95% CI 10.1–19.2), respectively. The median PFS differed significantly between EGFR exon 19 deletion and the L858R point mutation (18.0 versus 9.4 months, respectively; p = 0.006). The median overall survival had not yet been reached. Severe adverse events included grade 3 skin rash (15%), hypertension (17%), aspartate transaminase/alanine aminotransferase elevation (17%), proteinuria (7%), intracranial hemorrhage (2%), and grade 4 perforation of the digestive tract (2%). There were no treatment-related deaths. Conclusion: Gefitinib in combination with bevacizumab as first-line therapy seems to be a favorable and well-tolerated treatment for patients with advanced NSCLC with activating EGFR gene mutations, especially those with EGFR exon 19 deletion mutations, although the primary end point was not met because the lower limit of the CI was less than 40%.

Synergistic Effect of Olaparib with Combination of Cisplatin on PTEN-Deficient Lung Cancer Cells

PARP enzyme plays a key role in the cellular machinery responsible for DNA damage repair. PTEN is a tumor-suppressor gene deactivating PI3K downstream of EGFR signaling. We hypothesize that PTEN-deficient lung cancer cells suppressed DNA damage signaling and that the absence of PTEN can sensitize these cells to a concurrent treatment of a DNA-damaging agent (cisplatin) and a PARP inhibitor (olaparib). To investigate the effect of olaparib and cisplatin on PTEN-deficient lung tumors, two EGFR-mutant (deletion in exon19) non–small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild-type) and H1650 (PTEN loss), were used. We transfected intact PTEN gene into H1650 cells (H1650PTEN+) and knocked down PTEN expression in the PC-9 cells (PC-9PTEN−) using short hairpin RNA (shRNA). Combination of cisplatin with olaparib showed a synergistic effect in vitro according to the combination index in H1650 cells. Restoration of PTEN in the H1650 cells decreased sensitivity to the combination. Ablation of PTEN in PC-9 cells increased sensitivity to olaparib and cisplatin. We also examined the effectiveness of cisplatin and olaparib in a xenograft model using H1650 and PC-9PTEN− cells. The combination of cisplatin with olaparib was more effective than each agent individually. This effect was not observed in a xenograft model using H1650PTEN+ and PC-9 cells. Mechanistic investigations revealed that PTEN deficiency caused reductions in nuclear RAD51 and RPA focus formation and phosphorylated Chk1 and Mre11. Thus, genetic inactivation of PTEN led to the suppression of DNA repair. Mol Cancer Res; 11(2); 140–8. ©2012 AACR.